MiR-21-3p regulation FGFR1/FGF21/PPARγ pathway induces atrial brosis by targeting FGFR1 in diabetes

Background: A relationship between the abundance of epicardial adipose tissue (EAT) and the risk of atrial brosis and atrial brillation (AF) in diabetes mellitus (DM) has been reported. And previous studies have shown that MicroRNA-21 (miR-21) is a regulatory factor in atrial brosis and AF. The aim of this study was to examine the role of different subtypes of miR-21 in EAT browning and atrial brosis under hyperglycemia conditions. Methods: In vivo, C57BL/6 wild type (WT) and miR-21 knockout (KO) mice were used to establish the diabetic model by intraperitoneal injection of streptozotocin (STZ). In vitro, the EAT adipocytes from miR-21 KO mice were cultured and transfected with miR-21-3p mimic or miR-21-5p mimic and co-cultured with atrial broblasts in both HG or LG conditions. The browning of EAT and the brosis of broblasts were assessed by western blotting, immunouorescence, Masson staining, and ELISA. The gain- and loss-of-function experiments were used to identied broblast growth factor receptor 1 (FGFR1) as the target gene of miR-21-3p. Results: In patients with DM and/or AF, serum hsa-miR-21-3p, instead of hsa-miR-21-5p, was signicantly up-regulated. And miR-21 KO clearly ameliorated the atrial brosis in the diabetic mice. miR-21-3p as a key regulator that controls EAT browning and participates in atrial brosis under hyperglycemia conditions. Moreover, our gain- and loss-of-function experiments showed that FGFR1, as a direct target of miR-21-3p identied a regulatory pathway in EAT adipocytes. Conclusions: MiR-21-3p the process of atrial brosis. Mechanistically, down-regulation of miR–21–3p increased expression of the FGFR1, contributing to the activation of the browning transcriptional program via FGFR1/FGF21/PPARγ. Importantly, in addition to fat browning in EAT, anti-miR–21–3p-mediated benecial effects, including decreased inammatory factors, inhibited brosis-related gene expression in atrial broblast in co-culture model. interleukin (IL–6) chemoattractant protein (MCP–1) levels in co-culture supernatants were measured by ELISA assay (TNF-α: ab208348; IL–6: ab100712; MCP–1: USA) according to the manufacturer’s


Introduction
Diabetes mellitus (DM), despite many treatments, remains a major public health problem that currently affects more than 425 million people worldwide 1 . Due to the long-lasting rise of blood glucose, DM can cause many complications to affect the cardiovascular system, kidneys, retina, nervous system, etc 2 . Atrial brillation (AF), the most common arrhythmia in clinical practice, confers a major cause of morbidity and mortality due to an increased risk of abnormal hemodynamics and thromboembolism 3 . DM, whether type 1 or type 2, according to a large number of clinical studies [4][5][6] , has been shown to be a signi cant promoter for AF, with the mechanisms of atrial structural and electrical remodeling 7 . The extensive atrial brosis is a hallmark of AF and is considered to play an important role in both initiating and perpetuation AF under hyperglycemia conditions 7 .
Epicardial adipose tissue (EAT), a metabolically active visceral fat reservoir surrounding and in ltrating myocardium and vessels, is recognized as a source of pro-in ammatory mediators involved in the onset and development of various cardiovascular diseases (CVD), such as coronary artery disease (CAD) and arrhythmias 8,9 . And the increase in EAT volume or thickness is associated with DM and is correlated with left atrial enlargement, atrial brosis, abnormal electrical conduction between cardiomyocytes, and results in the onset of AF 10,11 .
As with other adipose tissue, EAT is also made up of both white adipose tissue (WAT) and brown adipose tissue (BAT) and presents the potential for trans-differentiation from BAT into WAT, or vice versa 12 .
Compared with less metabolically active WAT depots, BAT is characterized by an abundance of mitochondria, capillaries, and uncoupling protein-1 (UCP-1). BAT is thought to play a protective role against cardiometabolic dysfunction by virtue of its role in non-shivering thermogenesis 13 . And browning of WAT might be a novel approach for prevention or treatment in CVD 13 . To date, WAT browning is thought to be impaired in DM 14 . And the mechanisms of EAT browning and its role in DM-induced atrial brosis remains to be elucidated.
Fibroblast growth factor 21 (FGF21) has signi cant effects on energy balance, glucose metabolism, and lipid metabolism 15 . It was originally reported that FGF21 identi ed the liver as its main source 16 . In the subsequent studies, its role in inducing the browning of WAT is slowly emerging 17 . The biological roles of FGF21 are affected through downstream signaling pathways by binding to broblast growth factor receptors (FGFRs) and proliferator-activated receptor gamma (PPARγ) activation 18 . And FGFR1 has been shown to mediate the regulation of FGF21 in WAT.
MicroRNAs (miRNAs) are a class of endogenous, 20-22-nucleotide non-coding RNAs. Their main function is to regulate post-transcriptional regulation of target genes expression by binding to the 3' untranslated region (3'-UTR) 19 . MicroRNA-21 (miR-21) has been shown to be associated with atrial brosis in previous studies [20][21][22] . But most of its mechanisms focused on the damage of cardiomyocytes and the activation of broblasts. Few reports have shown the relationship between miR-21 and the browning of epicardial adipose tissue. Since miR-21 is abundantly expressed in EAT and FGFR1 has potential miR-21-3p-binding sites in the 3′UTR, in this work, we identify miR-21-3p (the subtype of miR-21 family) as a key regulator that controls EAT browning and participates atrial brosis under hyperglycemia conditions. Moreover, FGFR1, as a direct target of miR-21-3p, is decreased in these conditions and controls WAT to BAT differentiation trough FGFR1/FGF21/PPAPγ.

Animal experiments
All animal procedures were performed in accordance with the Guide of US National Institutes of Health for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Shanghai ninth people's hospital of Shanghai Jiaotong University School of Medicine, China. All mice used for these studies were on the C57BL/6 genetic background. Male wild-type and miR-21 knockout (KO) mice, purchased from Shanghai Model Organisms Center (Shanghai, China), aged 8 to 10 weeks, were used in the studies. Mice were assigned to one of four groups randomly: salinetreated WT group, STZ-treated WT group, saline-treated miR-21 KO group, and STZ-treated miR-21 KO group. WT mice and miR-21 KO mice were both infused with 150 mg/kg streptozotocin (STZ) or an equivalent volume of saline as a bolus intraperitoneally, and the blood glucose level was checked after 7 days. Mice with blood glucose levels of 16.7mM were considered to be diabetic and used for the experiments in STZ-treated groups, with each group contained 6 mice. The blood glucose levels of each group were shown in supplementary material Table S1 MiR-21 WT/KO mouse EAT pre-adipocytes culture and differentiation The mouse pre-adipocytes isolation was performed as previously described 23 . In brief, EAT was isolated under sterile conditions, washed twice in phosphate buffered solution (PBS) supplemented with 1% penicillin/streptomycin and then minced into 1 mm 3 pellets. Enzymatic digestion was performed with 2 mg/ml collagenase II (Worthington Biochemical, USA) at 37℃ in the water bath for 1 h with gentle agitation and terminated by the same volume of DMEM/F12 (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS). The suspension was ltered on a 75μm nylon cell strainer and centrifuged at 2000 r/min for 5 min. After disposing of the red blood cell lysed solution, the suspension was centrifuged at 2000 r/min for 5 min again and the pre-adipocytes were re-suspended in DMEM/F12 supplemented with 10% FBS and diluted to a nal concentration of 10 6 cells/ml. These cells were cultured at 37℃ in a humidi ed atmosphere containing 5% CO 2 . Hereafter, EAT pre-adipocytes isolated from WT mice and miR-21 KO mice were induced to differentiate into mature adipocytes as fellows when reached 80%con uence. Cells were incubated with 0.25 μM dexamethasone, 0.5 mM 3-Isobutyl-1-methylxanthine and 10μg/ml insulin for 3 days, then thoroughly rinsed with culture media and incubated for an additional 3-4 days with 10μg/ml insulin. Differentiation was con rmed by morphological changes, including intracellular lipid droplet accumulation, as con rmed by microscopic observation. Then mature adipocytes treated with low glucose (LG) (5.5 mM) and high glucose (HG) (30 mM) for 72 h.

Oil Red O Staining
Cultured adipocytes were washed with PBS and xed with 4% formaldehyde at room temperature for 15 min. Then the cells were stained with the Oil Red O working solutions containing 6 ml Oil Red O stock solution (5 g/l in isopropanol) and 4 ml ddH2O for 20 min. After staining, the cells were washed with 60% isopropanol in PBS and observed using an Olympus TH4-200 microscope (Japan). And the Oil Red signals were quanti ed by measuring the optical density at 490 nm.

Isolation and culture of adult mouse atrial broblasts
The experimental procedure to isolate adult mouse atrial broblasts was performed as we previously described 24 . In brief, the hearts from 8-10 weeks old male C57BL/6 mice were rapidly excised and submerged in cold phosphate buffered saline. The atria were cut into pieces with a tissue chopper. The pieces were washed and cells were dissociated with 1mg/ml collagenase II (Worthington Biochemical, USA). After 30min at 37°C, tissue pieces were discarded, and the supernatant, containing the isolated cells, was centrifuged at 1500 r/min for 5 min. Cells were resuspended in DMEM containing 10% FBS, and the resulting cell mixture was pre-plated in a 5% CO2 incubator at 37°C for 90min. After removal of the myocyte-enriched medium, DMEM was then added to the pre-plated broblasts, which were cultured for 3-4 days before being passaged. These cultured cells were also characterized as we previously described 24 .
Co-culture of atrial broblasts and adipocytes Atrial broblasts and EAT fully-differentiated adipocytes were co-cultured using transwell inserts with a 0.4-μm porous membrane, which separated the adipocytes (5.0×10 5 cells/well) in the lower chamber from the broblasts (5.0×10 5 cells/well) cells in the upper chamber. Each cell type was grown independently on the transwell plates. The hanging insert is constructed with a membrane having pores that are large enough to permit the passage of small molecules, yet small enough to prevent the passage of even the most motile of cell types. Following co-culture for 72 h after the incubation period, the biomarkers of adipocytes browning and brosis-related gene expressions were measured in adipocytes and broblasts, respectively. The co-culture of atrial broblasts and adipocytes were treated with low glucose (LG) (5.5 mM) and high glucose (HG) (30 mM) for 72 h in a humidi ed atmosphere containing 5% CO 2 at 37℃.
Histology, immunohistochemical staining, and immuno uorescence staining EAT or atrial tissues from mice were xed with 10% phosphate-buffered formalin for 24 h. Fixed tissues were then para n-embedded and serially sectioned with a microtome (4 μm thickness). The extent of interstitial brosis was evaluated by Masson staining. Immunohistochemical staining for collagen I, collagen III, alpha-smooth muscle actin (α-SMA), transforming growth factor-beta (TGF-β) and UCP-1 in atrial or EAT sections were also performed. Immuno uorescence staining of UCP-1 was performed in vitro cultured adipocytes. Images were acquired and analyzed by Image-Pro Plus 6.0.

RNA preparation and analysis
Total RNA was extracted from EAT and adipocytes using the TRIzol reagent (Invitrogen, USA). Reversetranscribed into cDNA using PrimeScript™ RT reagent Kit (Takara, Japan). Next, the cDNA was quantitatively ampli ed using TB Green Premix Ex Taq II (Takara, Japan). Real-time PCR was conducted in triplicate using an Applied Biosystems 6Flex. The sequences of the forward and reverse primers used for ampli cation are shown in supplementary material Table S2. The results for the expression of FGFR1 and UCP1 were presented relative to the expression of the GAPDH gene and relative miR-21 expression was normalized to the expression of U6 small nuclear RNA (snRNA) by the Delta-Delta Ct method.
Patients and serum miRNAs measurements

Results
MiR-21 participated in atrial brosis and EAT browning in mouse DM model As exhibited in gure 1, compared to the control group, DM induced a signi cant atrial interstitial collagen deposition, and this change was signi cantly attenuated by miR-21 KO (Figure 1a-1b). Besides, DM induced an increased expression of brosis-related proteins, such as TGF-β1, CTGF, Collagen I, collagen III and α-SMA in atrial tissue, which were also markedly alleviated by miR-21 KO (Figure 1c-1e). More interestingly, both the level of miR-21 and miR-21-3p were rise after STZ treatment in WAT of WT or miR-21 KO mice (Figure 2a-2b). Hyperglycemia conditions resulted in a decreased expression of WAT browning biomarker (UCP-1), which was signi cantly reversed in miR-21 KO mice (Figure 2c-2d).
Furthermore, we evaluated the WAT browning associated proteins and found that FGFR1, FGF21, and PPARγ expressions were decreased in diabetic model and were partly reversed by miR-21 KO (Figure 2e).
MiR-21-3p played a major role in browning of EAT We investigated the expression of different miR-21 subtypes in various clinic conditions, including healthy control, DM, AF, DM, and AF. Table 1 demonstrates the demographic and baseline characteristics of enrolled patients. And the result showed that the expression of serum hsa-miR-21-3p, instead of hsa-miR-21-5p, was signi cantly increased in patients with DM and/or AF (Figure 3a). The Oil Red staining of mature adipocytes was performed after differentiation induction of the EAT pre-adipocytes from WT or miR-21 KO mice ( Figure 3b). Hereafter, the result of PCR showed that miR-21-3p, rather than miR-21-5p, was signi cantly increased in mature adipocytes differentiated from EAT pre-adipocytes of WT mice after glucose treated (Figure 3c). For further proof, the isolated EAT pre-adipocytes from miR-21 KO mice transfected with mimics NC, miR-21-3p mimics or miR-21-5p mimics to achieve the overexpression of the corresponding miR-21 subtype (Figure 3d) and then induced to differentiate into mature adipocytes. The results of Western blotting and immuno uorescence showed that UCP-1 expression was signi cantly inhibited by miR-21-3p mimics rather than miR-21-5p mimics transfection (Figure 3e-3f) which indicated that miR-21-3p was involved in EAT browning.
MiR-21-3p inhibited browning of EAT to promote hyperglycemia-induced atrial brosis EAT pre-adipocytes from miR-21 KO mice transfected with mimics NC, miR-21-3p mimics or miR-21-5p mimics, and then induced to differentiate into mature adipocytes. Full-differentiated adipocytes cocultured with atrial broblasts from WT mice and were treated with low glucose (LG) (5.5 mM) and high glucose (HG) (30 mM) for 72 h. We observed that hyperglycemia conditions induced a signi cantly higher expression of brosis-related proteins in broblasts (Figure 4a-4b).
Besides, under the same hyperglycemic conditions, miR-21-3p transfected adipocytes resulted in an up-regulated expression of brotic proteins in broblasts compared with mimics NC or miR-21-5p mimics transfected adipocytes (Figure 4a-4b). We also determined the in ammatory factor in co-culture supernatants, including TNFα, IL-6, and MCP-1, and the results indicated that these in ammatory factors were markedly increased by miR-21-3p mimics transfection into adipocytes instead of mimics NC or miR-21-5p mimics in both LG and HG conditions (Figure 4c). At last, the same trend in WAT browning biomarker, UCP1 expression, was also observed (Figure 4d-4e). The aforementioned results indicated that miR-21-3p might be involved in EAT browning and participate in hyperglycemia-induced atrial brosis. At the same time, EAT preadipocytes from miR-21 WT mice transfected with mimics NC, miR-21-3p mimics were implemented which showed the same result in corresponding experiments ( Figure S1).
Western blotting was also used to analyze FGFR1, FGF21, PPARγ expression of adipocytes in our coculture model. If miR-21-3p mimic transfection into adipocytes, these EAT browning associated proteins were e ciently inhibited in adipocytes in HG condition compared with LG condition. Furthermore, under the same HG condition, miR-21-3p mimic transfection into adipocytes resulted in lower expressions of WAT browning associated proteins compared with mimics NC and pcDNA-FGFR1 transfected could partially reverse this phenomenon (Figure 6c). Three in ammatory factors (TNFα, IL-6, and MCP-1) were markedly increased by miR-21-3p mimic transfection compared with mimics NC in both LG and HG conditions (Figure 6d) and pcDNA-FGFR1 transfected could partially reverse this phenomenon. Finally, the WAT browning biomarker, UCP1, was markedly inhibited by miR-21-3p mimic and pcDNA-FGFR1 transfected could partially reverse this phenomenon (Figure 6e-6f).

Discussion
In the current study, we further identi ed a regulatory pathway in EAT adipocytes consisting of miR-21-3p, FGFR1, FGF21 and PPARγ that control EAT browning and participates the process of hyperglycemiainduced atrial brosis. Modulation of this signaling pathway might provide a therapeutic option for the prevention and treatment of atrial brosis in DM.
Numerous evidence has suggested that diabetes is a strong, independent risk factor for AF 7,27 . The underlying mechanisms by which diabetes increases the susceptibility to AF are unclear but are thought to be associated with electrical and structural remodeling of the atria 28,29 . Extensive atrial brosis resulting from hyperglycemia is thought to play a key role in both initiating and perpetuation AF as an increase of collagen deposition in the atria can cause abnormal conduction of signals, rupture of the propagating waves, and even the occurrence of re-entry 30 . In diabetic conditions, broblasts are activated leading to inappropriate collagen production and deposition no matter in type I DM 31,32 or type II DM [33][34][35] . Emerging evidence poses a unique challenge to understanding the pathogenesis of atrial brillation caused by diabetes, which is often consistent with obesity. Obesity leads to increased thickness of EAT and enhanced invasiveness, which in turn leads to interstitial brosis. Recent studies have reported a higher volume or thickness of EAT in patients with AF, particularly in those with non-paroxysmal AF [36][37][38] . Some studies have reported a relationship between left atrium size and EAT thickness or volume, which might contribute to atrial brosis or cardiomyocytes electrophysiological disorders leading to AF 10,11,37 . In particular, posterior left atrial adipose tissue is supposed to contribute to the atrial remodeling leading to the onset of AF 39 . Moreover, under diabetic or hyperglycemic conditions, high volume of thickness of EAT is also observed [40][41][42] . There are several possible mechanisms for the associations between EAT and the increased risks of AF. First, EAT is a rich source of adipokines and cytokines which have pro-brotic and pro-in ammatory effects, and the closed proximity of EAT to atrial cardiomyocytes might favor the paracrine activity of EAT secretome, which seems to play a role in the pathogenesis of AF 10 . Second, adipocyte in ltration within atrial cardiomyocytes might lead to the loss of side-to-side cell connection with consequent reduced and heterogeneous voltage [43][44][45] . Third, the brotic remodeling of EAT was also associated with atrial myocardial brosis 10 .
On the other hand, EAT is also made up of WAT and BAT. BAT is the primary site of non-shivering thermogenesis and is, therefore, a relevant site for adaptive energy expenditure processes. Compared with WAT, BAT improves insulin sensitivity, glucose tolerance, lipid homeostasis, and protects against the pathogenesis of CVD 13,46 . It has been recently shown that adipose tissues have remarkable plasticity in relation to their contents of white and brown adipocytes. Modulation of the cardiac and vascular adipose tissue to increase the proportion of thermogenic brown or beige adipocytes might be a viable way to improve local in ammation and reduce cardiovascular risk 13,46 . However, under diabetic or hyperglycemic conditions, impaired WAT browning potential is observed 14 , which might aggregate the pathogenesis of AF. Consistent with previous studies, our experiments indicated that hyperglycemia inhibited the biomarkers of BAT in mouse EAT as well as in vitro cultured adipocytes, suggesting that hyperglycemia might decrease the process of EAT browning.
To date, the mechanisms of EAT browning and its role in DM-induced atrial brosis remain to be elucidated. FGF21 has been shown to have a bene cial effect on metabolism and energy balance by enhancing fatty acid-oxidation during prolonged fasting and also by promoting WAT browning [47][48][49][50] . And a recent clinical study showed that cold exposure increased circulating levels of the fat browning activators FGF21 and irisin and that treatment with either of these endocrine regulators up-regulated browning genes and promoted thermogenesis 51 . Mechanistically, FGF21 activates cell signaling by binding to a heteromeric cell-surface receptor tyrosine kinase complex composed of β-Klotho and FGFR1 49 . Both β-Klotho and FGFR1 are abundantly expressed in WAT, where FGF21-regulated genes are involved in a variety of metabolic processes including lipogenesis, lipolysis, fatty acid oxidation, and WAT browning 49 . Furthermore, PPARγ, a member of the nuclear receptor family of ligand-activated transcription factors, is also required for adipocyte differentiation. PPARγ agonist has been shown to induce browning of the EAT that probably contributes to the increase in lipid turnover 52 . And FGF21 was thought to be a key mediator of the physiologic and pharmacologic actions of PPARγ in WAT 49 . FGF21 stimulates PPARγ transcriptional activity and FGF21 de ciency mice have decreased PPARγ activity in WAT and corresponding reductions in WAT mass and adipocyte size 49 . On the other hand, FGF21 was previously shown to be induced by PPARγ agonists in WAT and to cooperate with rosiglitazone in promoting differentiation in 3T3-L1 adipocytes 53,54 . And obese, insulin-resistant mice lacking FGF21 are refractory to the actions of rosiglitazone, including both bene cial and adverse effects 49 . Therefore, we conclude that the actions of FGF21 and PPARγ are fundamentally intertwined, and propose a feedforward regulatory model in WAT 55 . And FGFR1/FGF21/PPARγ might be an important signal pathway to precipitate the browning of WAT. In our mouse diabetic model as well as in vitro cellular model, we found that FGFR1/FGF21/PPARγ pathway was inhibited in EAT or under hyperglycemia conditions. Consistently, FGF21 was also shown to protect the blood-brain barrier through FGFR1/FGF21/PPARγ activation, which up-regulated tight junction and adhesion junction proteins 56 .
Recent studies indicated that miRs function as important regulators that participates in DM-induced atrial brosis 35 . The role of miR-21 in the pathogenesis of atrial brosis has been illuminated, and increased miR-21 expression was correlated positively with atrial brosis or brotic gene expression [20][21][22] . Many target genes of miR-21 have been found to play a large role in DM-induced atrial brosis through different biological pathways. Tao H et al. found that miR-21 regulated atrial brosis via dysregulation of WW Domain-Containing Protein 1 57 . Cao W et al demonstrated that the tumor suppressor cell adhesion molecule 1 was the potential target of miR-21, and miR-21 promoted cardiac brosis via STAT3 signaling pathway by decrease CADM1 expression 58 . In our mice diabetic model, we also found that miR-21 KO manifested a decreased atrial brosis as well as brotic gene expression. However, miR-21 has several subtypes, such as miR-21-3p and miR-21-5p in human, and miR-21-3p and miR-21-5p in mice. We further investigated the expression of different miR-21 subtypes in various clinic conditions, including healthy control, DM, AF, DM combined AF. And the results indicated that hsa-miR-21-3p, instead of hsa-miR-21-5p, was obviously increased in patients with DM and/or AF, suggesting that hsa-miR-21-3p is more likely to be involved in the regulation of DM-induced atrial brosis. Besides, emerging evidence indicates that miRs also function as important regulators in brown remodeling of adipocytes.
Muscle-enriched miR-133a directly down-regulated expression of the key transcriptional activator of brown fat differentiation, positive regulatory domain containing 16 (PRDM16), and cold exposure decreased miR-133a levels and promoted brown fat cell differentiation 59,60 . Brown adipocyte-enriched miR-155 was also shown to inhibit brown fat cell differentiation by directly targeting the browning transcription factor C/EBP 61 . But whether miR-21-3p played a role in the browning of EAT under diabetes and thus affected atrial brosis has not been studied. In the present study, we further identify miR-21-3p as a key regulator that controls EAT browning in hyperglycemia condition. Further, we predicted target genes by Targetscan and found browning transcription factor FGFR1 as its potential target. Besides, by virtue of in vitro co-culture model, miR-21-3p regulated brotic gene expression in atrial broblasts through affecting the adipocytes browning.
A paracrine effect of EAT on the neighboring myocardium has been proposed 10 . EAT produces a number of in ammatory mediators and adipocytokines that can modulate the functional and structural properties of the myocardium 62,63 . In this line, it has been shown that the secretome of EAT can induce atrial brosis, an important determinant of the substrate of AF. In the present study, we observed that miR-21-3p mimics for adipocytes could increase the levels of in ammatory factors in co-culture model in hyperglycemia conditions.
Our current study has some limitations that deserve to be mentioned. First, mouse adipocyte-speci c miR-21-3p KO was not performed in this study. Second, we did not conduct in vivo intervention experiments to study the correlation between miR-21-3p and EAT browning. Third, we only determined the extent of atrial brosis, however, we did not perform the experiments of cardiac electrophysiology and programmed stimulation, such as inducibility of AF.

Conclusions
In this study, we showed that miR-21-3p under hyperglycemia conditions act as an inhibitor of EAT browning and participated in the process of atrial brosis. Mechanistically, we showed that downregulation of miR-21-3p increased expression of the FGFR1, contributing to the activation of the browning transcriptional program via FGFR1/FGF21/PPARγ. Importantly, in addition to fat browning in EAT, anti-miR-21-3p-mediated bene cial effects, including decreased in ammatory factors, inhibited brosis-related gene expression in atrial broblast in co-culture model.

Disclosures
The authors have no con icts of interest to disclose.  Browning of pericardial adipose tissue were analyzed 3 months after the modeling in WT and miR-21 KO mice (n=6). a and b. The relative miR-21 and miR-21-3p expression were detected by qRT-PCR in pericardial adipose tissue. c. Western blotting was used to analyze UCP1 expression and quantitative by Image J. d. UCP1 expression was analyzed by immunohistochemistry (×400; scale bar, 50 µm). e.

Figure 3
MiR-21-3p plays a major role in browning of white fat. a. The relative levels of hsa-miR-21-3p and hsa-miR-21-5p in the serum of different patients were detected by qRT-PCR (n=20). b. Adipocytic differentiation of pre-adipocytes isolated from miR-21 KO mice and Oil Red O staining of mature adipocytes after differentiation induction (×400, scale bar, 50 µm). c. The relative miR-21-3p and miR-21-5p mRNA expression in pre-adipocytes extracted from WT mice after treated with low glucose (LG) (